Method and test kits for detection of bacteriophage

ABSTRACT

A method for detection, identification and/or quantification of bacteriophage of bacterial host specificity for bacterial genus, species or serotype, based upon the occurrence of release of cell contents, particularly nucleotides e.g. ATP, on lysis of bacterial cell walls on incubation with bacterial host cells. When new phage particles are released at the end of the phage replication cycle nucleotide levels are measured and compared with controls. The method provides for the detection of specific phages which is faster and more sensitive than known techniques. The method is only limited by the availability of host bacteria/target phage pairings.

The present invention relates to a method of detection, identificationand/or quantification of bacteriophage (phage or phages), and to testkits for use in carrying out that method. Particularly the methodenables detection of bacteriophages specific to particular bacterialgenus, species or serotype, whether in isolated form or as contaminantsin environmental or forensic samples, or in foodstuffs.

Although often undesirable, bacteria also have many industrialapplications. Increasingly important is the role of bacteria in the areaof genetic engineering and particularly in the large scale production ofprotein products as genetically engineered bacteria. More traditionallythey have been used in production of natural products in fermentation,notably in production of cheese and milk fermentation products. Therapid fermentation of lactose to lactic acid is the principal reactionin the manufacture of such milk derived products and is initiated by theaddition of starter cultures of species of lactic acid bacteria to themilk substrate. On occasion, for various reasons, these starter culturesfail and normal acid development fails to begin or is not maintained.

One of the most important reasons for starter culture failure is thepresence in the milk of bacteriophage, often originating from the dairyenvironment. These are specific viral agents that attack and killbacteria, in this case those of the starter culture. The bacteriophagereproduce parasitically in their bacterial hosts, resulting in a progenyof new phage particles which are liberated into the environment uponlysis of the bacterial cells. A bacteriophage infection in a dairy plantresults in a serious decrease and, in some cases, a total failure in theproduction of lactic acid by the starter cultures. For many years theproblem of phage has been the most serious one confronting thecheesemaker because of the economic losses it entails. These include thetime lost in manufacture, loss of the raw material and of substandardproduct (see Klaenhammer (1984) Adv. Appl. Microbiol. 46, 313-25 andHeap & Lawrence (1988) Developments in Food Microbiology, Elsevier).Conventional techniques used in the dairy industry for bacteriophagedetection are based upon traditional microbiological technology, and areboth labour intensive and time consuming. These include observation ofbacterial plaques, ie. clear areas in a background of bacteria, producedon lawns of starter culture bacteria in petri dishes and measuring therate of lactic acid production in cultures, both inoculated withenvironmental samples. It is clear that the development of a rapid yetsimple bacteriophage detection technique would be of immense benefit tothe industry.

The present inventor has now provided such a technique, which in itspreferred form uses bioluminescent techniques to provide a light signalindicative of the presence and amount of a specific bacteriophage orbacteriophage type in a sample under investigation, giving a positiveresult within about 4 to 5 hours, as opposed to 24 hours or more forexisting methods.

The method of the invention is based upon the release of cellularcomponents from bacteria infected with bacteriophage, particularly whenthe bacteriophage undergoes lytic cycle replication. In this cycle thebacteriophage take over the metabolism of the cell and replicatethemselves, whereby at the end of the cycle the bacterial cell wallsrupture to release progeny and substantially the entire bacterial cellcontents. By measuring one or more particular components foundassociated with the bacterial cell that are made accessible to reagentsin an incubation medium by the phage it is possible to measureinfection, eg. via lysis, and using calibration curves or otherstatistical techniques, it is thus possible to estimate the amount ofphage in the original sample. By exposing the sample to a bacteria thatis a specific target of the phage which is the subject of a test, andproviding incubation conditions that would allow infection of thatbacteria by the specific phage, it is possible to determine presence andamount of specific phage, even in the presence of those that do notcause infection, by assaying for particular cellular components madeaccessible as described above.

The present invention thus provides a method for the detection,identification and/or quantification of target bacteriophage ofpredetermined bacterial host specificity in a material underinvestigation comprising incubating a sample derived from the materialwith bacteria of that host type, preferably in a liquid culture, underconditions such that they are caused to release cellular components ifinfected with the target phage, measuring the amount of one or moreparticular components released from the bacteria by any bacteriophagepresent in the sample during the incubation, and relating this to thepresence, identity and/or amount of target bacteriophage. Preferably theparticular component or components comprise nucleotides.

It is theoretically possible to measure any of the nucleotides that arereleased by the cell lysis caused by the release of new phage particles,for example NAD, NADP. NADH, NADPH, ATP or ADP, cAMP or cGMP, withsensitivity provided by use of one or more of the many enzyme basedassay systems, eg. `cascade` systems, that are available in the art. Forexample, GB 2213261 discloses a method which may be used for assayingreduced pyridine nucleotides, eg NADH or NADPH, based upon a salicylatemonooxygenase system, while other enzyme systems such as the alkalinephosphatase (EC 3.1.3.1)/NAD/NADP system as disclosed in GB 2240845.Suitable assay systems for ADP, cAMP, cGMP etc will occur to thoseskilled in the art.

However, particularly preferred is the measurement of adenosinetriphosphate (ATP), that being readily measurable by assay with avariety of enzyme/enzyme substrate combinations by virtue of its being acofactor in numerous substrate conversions, and being released inrelatively large quantities as compared with other bacterialnucleotides. For the rapid and efficient determination of levels ofreleased ATP in the present application it is especially preferred toutilise enzymes which result in the production of luminescence, mostconveniently the enzyme luciferase. ATP release is quantifiable withcommercially available reagents using the process of bioluminescencewherein it is used to drive the reaction in which luciferase catalysesthe oxidation of luciferin resulting in the emission of light. Thequantum efficiency of this reaction is extremely high and the amount oflight produced gives a measure of the amount of AWP originally presentin the sample before the assay reaction depleted it.

For identification or quantification of specific phage occuring inmaterial in relatively high concentrations, eg. in cultures of isolatedphage, it is possible merely to incubate the specific host bacteria witha sample of the material in the presence of, or with subsequent additionof, the component assay reagents and thus to measure the amount ofcomponent released by performing the assay.

Where lytic phages are being targeted, at the end of the replicationcycle the specific host bacteria infected by the target bacteriophageburst (times range from eg. 20-60 minutes depending on species),cellular component, eg nucleotide, is released and detected by theassay. Control samples either containing no phage or phage withoutbacteria show no increase in levels above background levels.

For identification, detection and/or quantification of specific phage atlower concentrations, for example as contaminants in or on water orfoodstuff materials, it is necessary to first perform an enrichment ofthe sample under test, eg. for a few hours, to allow the target phage tomultiply to a level where released components. eg. nucleotides, will bedetectable above background levels. This enrichment is preferablycarried out by inoculating using a culture, preferably a log phaseculture, of further host bacteria of type capable of being parasitisedby the target bacteriophage, preferably specifically.

After the incubations with the host bacteria to increase phage count asample of cell free phage enriched medium is mixed with a culture of thestarter organisms, preferably in log phase, and incubated for a settemperature for a set time selected to cause release of cell component,these being dependent upon the characteristics of the phage/bacteriacombination being used and the component, eg. nucleotide, whose releaseis to be measured using an appropriate assay, eg. enzyme/substratesystem.

The sample to be analysed should preferably be filtered to remove anybacteria before being added to the enrichment or component release step,thus avoiding possible interfering release due to the action ofnon-target bacteriophage on contaminating bacteria. For the purpose oftaking samples from an environment under investigation, conventionaltechniques may be used such as swabbing, eg. of surfaces, or samplingaliquots of liquids.

Dependent upon the biology of the phage/host pair selected, the methodhas the potential for great specificity and still further sensitivityand rapidity. Utilising the aforesaid ATP assay method the inventor hasreadily developed systems capable of the detection of phage specific toStaphylococcus aureus, Listeria monocytogenes, Salmonella, E. coli andpseudomonads. Those skilled in the art will appreciate that there is nolimit to the application of the present method other than theavailability of necessary specific phage/bacteria pairings. For thepreferred luminescence assay method whereby ATP release is measured thetest sample, whether enriched or not for phage, is conveniently mixedwith the bacterial culture, preferably as log phase, in a luminometertube and incubated for a suitable time. This will commonly be from 30 to60 minutes depending on the phage/bacteria system. After or over thisperiod, preferably after, the released ATP is measured using a lightproducing assay, eg. the luciferin/luciferase reaction system, toproduce an amount of light which is detected in a luminometer andrelated to the amount of ATP.

In all cases controls may advantageously be carried out for comparisonof background component levels, eg. nucleotide levels, eg. in samples oflog phase culture medium without the bacteria and/or incubated withbacteria and a known amount of the phage, thereby enabling production ofcalibration curves. Such controls may include challenge with other hostsof different specificity for determining more completely thecharacteristics or various types of phage present in the sample.Similarly several bacterial types may be used in one incubation wherethe method is being used to screen for a number of types of phage forwhich no one common bacteria is specific enough.

The range of bacteria available and the bacteriophage for which they arespecific will be realised to be vast by those skilled in the art. Forexample a list of phage types available from the American Type CultureCollection (ATCC) is published by them as the `Catalogue of Bacteria &Bacteriophages`. Other such depositories also publish equivalent data intheir catalogues and this may be used to identify possible phage`reagents` for the present method. Bacteria may be used, inter alia, inaqueous suspension or in freeze dried form eg. on microtitre platewells. In this manner plate luminometry can be used. The method of theinvention is particularly intended for use to detect and quantify lyticphages, ie. those that lead to lysis of the test bacteria, but any phagethat causes release of cellular components by its actions may bedetected.

The present invention also provides test kits for carrying out themethod of the present invention and these are characterised in so far asthey comprise a bacteria selected for the ability to be infectedspecifically by target bacteriophage, ie. a type or types for whichdetection, identification and/or quantification is desired, incombination with some or all of the reagents which are specificallyassociated with the aforesaid method of the invention.

Thus preferably test kits of the present invention comprise

(a) a bacteria selected for its ability to be specifically infected bytarget bacteriophage and caused to release cellular components thereby,and at least one of

(b) the reagents necessary for carrying out assay for cellular componentreleased by action of the bacteriophage on the bacteria and

(c) a bacteria for supporting growth of target bacteriophage.

Thus preferred test kits of the invention are those wherein the reagentsnecessary for carrying out assay are for assay of the amount of anucleotide released by the bacteria, preferably reagents comprisingluciferin and luciferase. Test kits optimised for performance of highsensitivity assay of bacteriophage will include bacteria of component(c) above, which may be the same as those of component (a), but may alsobe less specifically parasitized bacteria which nevertheless are capableof supporting more rapid replication of phage or producing a higherquantity of phage.

The method and kits of the present invention will now be exemplified byway of illustration only by reference to the following non-limitingexamples. The vast variety of options available will be readilydeterminable by those skilled in the art on consideration of the generalmethod described above and particularised below, and the available typesof bacteria/phage pairings and eg. nucleotide assays.

EXAMPLE 1

Kit for Use in the Detection of Bacteriphages Specific for BacterialStarter Cultures in the Dairy Environment and in Dairy Products.

A kit for use in the method of the invention conveniently comprises theitems marked with an asterisk below and optionally supplemented with anyof the other equipment and reagents set out below as required for themethod of the Example.

Equipment required: Sterile cotton swabs; 0.8 μm pore-size syringefilters; 0.22 μm pore-size syringe filters; sterile 5 ml syringes;sterile universal bottles or bijou bottles; centrifuge tubes;luminometer (model LB953 Autolumat; Berthold Instruments UK Ltd, StAlbans, Hertfordshire); polystyrene luminometer tubes (Sarstedt.Beaumont Leys, Leicester).

Reagents required: Sterile peptone/saline (1 g per liter/8.5 g per literdistilled water respectively); M17 broth (Unipath Limited, Basingstoke),*Adenosine-5'-triphosphate assay mix containing luciferin/luciferase anddilution buffer (Sigma Chemical Company Limited, Poole, Dorset); sterile10% lactic acid; *stock culture of starter culture bacteria (SeeBulletin of the IDF 263/1991 Chapter 2).

EXAMPLE 2

Method for Detection of Bateriophages Specific for Bacterial StarterCultures (A) in the Dairy Environment and (B) in Dairy Products,

A: Dairy equipment method:

Sampling: A sterile cotton swab dampened in sterile distilled water wasused to swab an area of equipment (0.5 square meters) and then agitatedin 5 ml sterile peptone/saline. The peptone/saline was then filteredthrough a 0.8 μm pore-size syringe filter into a sterile container, thenfiltered again through a 0.22 μm pore-size syringe filter into a secondsuch container. If bacteriophage liter was anticipated as being low aphage enrichment step was carried out.

Enrichment: 8 ml of fresh M17 broth was inoculated with 1 ml oflog-phase culture of the starter organisms, 1 ml of the bacteriophagefiltrate was added and the mix incubated at a suitable temperature for 4hours (20-22° C. for mesophilic starter cultures and 42° C. forthermophilic starter cultures). This enriched culture was transferred toa centrifuge tube and spun to remove bacteria (5000 g for 20 minutes),before the supernatant was filtered through a 0.22 μm pore-size syringefilter into a sterile vessel.

Assay: 50 μl of M17 broth, held at between 22° C. or 42° C. depending onhost starter culture used as set out above, was added to all 80polystyrene luminometer tubes in the luminometer, prewarmed to the sametemperature. 50 μl of a log-phase starter culture was placed into eachalternate tube to act as negative control, and 0.5 ml of `phage`filtrate from the enrichment or the sampling step was added to 4.5 ml ofthe remaining culture and 50 μl of this placed in each of the remainingluminometer tubes. Light measurement was commenced with 100 μlluciferin/luciferase reagent injected into each tube and the lightoutput measured over periods of 60 seconds. Peak light measurements(counts per second) were then plotted against time for both sample andnegative control tubes.

Result: An approximate 5 fold increase in free ATP levels of the testsamples against the negative controls were shown to indicate presence ofstarter-specific phages in the test sample.

B: Dairy product method:

Sampling: Liquids required no particular pretreatment; powdersadvantageously were suspended in an appropriate volume (eg. 1:10dilution) of sterile water; solids such as cheese were mixed with ninetimes their weight of peptone/saline then homogenised. The pH of allsamples was adjusted aseptically to 4.5-4.7 to precipitate casein. 0.3ml sterile 10% lactic acid was added to 10 ml milk or whey; pH electrodeuse was avoided unless autoclaved due to risk of contamination. Thesamples were centrifuged at 5000 g for 20 minutes and the supernatantfiltered through a 0.22 μm pore-size syringe filter into a sterilecontainer to remove large particles and bacteria. As in (A) above, thebacteriophage titre in this sterile filtrate was increased by anenrichment step if suspected to be low. Enrichment and light measurmentsteps were performed as described in Method A above.

I claim:
 1. A method for the detection, identification and/orquantification of target bacteriophage of predetermined bacterial hostspecificity in a sample under investigation, said method comprising thesuccessive steps of:(a) enriching the bacteriophage present in thesample by incubating the sample with a culture of host bacteria capableof being parasitised by the target bacteriophage wherein it is allowedto multiply; (b) incubating the enriched sample with bacteria of thathost type in the presence of or subsequently adding an enzyme assayreagent under conditions selected such that the bacteria are caused torelease cellular components if infected with the target bacteriophage;(c) measuring the amount of one or more particular components releasedfrom the bacteria by any bacteriophage present in the sample during theincubation; and (d) relating the value measured in step (c) to thepresence, identity and/or amount of target bacteriophage.
 2. A method asclaimed in claim 1 wherein the particular cellular component orcomponents measured comprise one or more nucleotides.
 3. A method asclaimed in claim 2 wherein the component or components measured are oneor more of NAD, NADP, NADH, NADPH, ATP or ADP, cAMP or cGMP.
 4. A methodas claimed in claim 2 wherein the nucleotide is measured using an enzymebased assay system.
 5. A method as claimed in claim 4 wherein the assaysystem is an enzyme cascade assay.
 6. A method as claimed in claim 1wherein the component is ATP and the assay is one that uses an enzymicreaction resulting in luminescence.
 7. A method as claimed in claim 6wherein the assay system is the luciferin/luciferase system.
 8. A methodas claimed in claim 1 wherein the luciferin/luciferase reaction reagentsare used and the amount of light given off is measured during and/or atthe end of the incubation.
 9. The method as claimed in claim 1 whereinthe enrichment incubation of step (a) is carried out for 1 to 4 hours.10. The method as claimed in claim 1 wherein the enrichment step of step(a) is carried out by adding the sample to a log phase culture of hostbacteria.
 11. The method as claimed in claim 1 wherein the enrichmentand component release incubations of steps (a) and (b) are carried outusing a number of different specificity host bacteria types whereby therelease of component is indicative of a number of bacteriophage types.12. A method as claimed in claim 1 wherein the sample to be analyzed isfiltered to remove any bacteria before being added to the enrichment orcomponent release steps.